S152: SETD2/H3K36ME3 DEFICIENCY SUSTAINS GENOMIC INSTABILITY AND ENHANCES CLONOGENIC POTENTIAL OF CHRONIC MYELOID LEUKEMIA (CML) PROGENITORS
Authors: M. Mancini, S. De Santis, C. Monaldi, S. Bruno, F. Castagnetti, G. Gugliotta, A. Iurlo, M. Cerrano, S. Galimberti, S. Balducci, F. Stagno, G. Rosti, M. Cavo, S. Soverini
Journal: HemaSphere (2022)

Background: Genomic instability is a hallmark of chronic myeloid leukemia (CML) cells since the chronic phase (CP) of the disease and results in BCR-ABL1 mutations and/or additional genetic and genomic aberrations that may drive resistance to tyrosine kinase inhibitors (TKIs) and progression to blast crisis (BC). Genomic instability is also a feature of CML stem cells and may underlie their persistence. We have recently reported that virtually all CML patients (pts) in BC display loss of function of SETD2, an enzyme that trimethylates histone H3 lysine 36 (H3K36me3). SETD2 is a tumor suppressor implicated in several neoplastic conditions. H3K36me3 is required for homologous recombination (HR) repair of double strand breaks and for DNA mismatch repair (MMR). Aims: We investigated SETD2/H3K36me3 status in CD34+ progenitors of CP CML pts and whether SETD2/H3K36me3 deficiency may play a role in genomic instability in CML models. Methods: CD34+ progenitor cells were isolated from 20 newly diagnosed CP CML pts and screened for SETD2 and H3K36me3 by Western blotting (WB). SETD2-proficient (LAMA84) and -deficient (KCL22) CML cell lines were also studied. SETD2 knock-down and SETD2 forced expression in CD34+ progenitors and cell lines were performed by RNAi and nucleofection, respectively. DNA damage and DNA repair activation were assessed by WB and immunofluorescence (IF). Clonogenic capacity was evaluated by clonogenic assays. Results: Loss of SETD2 protein expression and function (the latter assessed using loss of H3K36me3 as surrogate marker) were detected by WB in the CD34+ cell fraction of 20/20 newly diagnosed CP CML pts, as compared to the corresponding total mononuclear cell fraction or to the CD34+ fraction obtained from a pool of healthy donors. To investigate whether SETD2/H3K36me3 loss impinges on the activation and proficiency of HR, we used UV rays to induce DNA damage in SETD2 siRNA-depleted LAMA 84 (SETD2-proficient) cells. Compared to control cells, cells silenced for SETD2 displayed an increase in the expression of the DNA damage maker γH2AX associated with a loss of RAD51 (HR) and MSH6 (MMR) repair foci. To confirm the role of SETD2 as a tumor suppressor implicated in maintaining genomic stability in CML, we transfected KCL22 (SETD2-deficient) cells with an ectopic SETD2 plasmid. SETD2 forced expression induced more than 50% reduction in cell doubling time and a significant reduction in clonogenic potential. Moreover, SETD2 overexpression was able to restore DNA damage response, as demonstrated by WB and immunofluorescence detection of H2AX phosphorylation, RAD51 (HR) foci, THEX1 (DNA replication) and MSH6 (MMR) observed after UV exposure. In line with the effects of SETD2 deficiency/overexpression observed in cell line models, CML progenitor cells displaying SETD2/H3K36me3 deficiency showed hyper-phosphorylation of H2AX and the overexpression of RAD51 (HR) and MSH6 (MMR) did not result in DNA repair foci. Moreover, forced overexpression of SETD2 restored proliferation control in CD34+ cells from 5 CP CML pts, since after nucleofection clonogenic assays showed a >50% reduction in clonogenic potential. Summary/Conclusion: SETD2/H3K36me3 deficiency is a novel, BCR-ABL1-independent mechanism of genetic instability in CML. SETD2 behaves like a true tumor suppressor in CD34+ progenitor cells of CP CML pts. Supported by AIRC IG 2019 grant (23001) and Italian Ministry of Health, “Bando Ricerca Finalizzata 2016”, project GR-2016-02364880.